用于研究RHD等位基因的有核红细胞体外表达系统的建立

Establishment of a erythroblast expression system in vitro for RHD allele studies

目的建立用于研究RHD等位基因的有核红细胞体外表达系统。方法从Rh阴性献血者的外周血中分离单个核细胞,并从中进一步分离、培养有核红细胞,同时使用含有野生型RHD等位基因和绿色荧光蛋白的慢病毒表达质粒和包装质粒共转染HEK 293细胞包装慢病毒,收获包装病毒颗粒后感染体外培养的Rh D阴性有核红细胞,诱导有核红细胞分化,最后使用针对9种不同D抗原表位的15种单克隆抗体对红细胞Rh D抗原表位进行流式分析。结果用携带野生型RHD基因的慢病毒感染体外培养的Rh D阴性有核红细胞,成功表达了抗原表位完整的D抗原,Rh D阴性有核红细胞的感染效率为51. 7%。结论成功建立了用于研究RHD等位基因的有核红细胞体外表达系统,可进一步应用于弱D、部分D和D放散型相关的RHD突变型等位基因的体外表达研究。

Objective To establish an in vitro erythroblast expression system for studying the effect of RHD alleles on the expression of Rh D epitopes. Methods The erythroblasts from donors with D-phenotype were separated from the peripheral blood and cultured in a expansion medium. Meanwhile,the lentivirus carrying the wild-type RHD allele was prepared by transfecting HEK 293 cells with RHD lentiviral construct and helper plasmids. The D-erythroblasts were then transduced with the wild-type RHD construct by lentiviral methods. After 48 hours transduction,the cells differentiated and tested for Rh D epitope expression by flow cytometry using 15 kinds of monoclonal anti-D reagents coupling with 9 epitopes of the D antigen.Results The D-erythroblasts transduced with the wild-type RHD construct showed a normal expression of D epitopes tested by flow cytometry. The infection efficiency of D-erythroblasts was 51. 7%. Conclusion The in vitro culture erythroblast expression system,which can be used to investigate the Rh D epitope expression of RHD alleles related to weak D,partial D and Del,was successfully established.